To find lead compounds for drug discovery programs, large numbers of compounds are often screened for their activity as enzyme inhibitors or receptor agonists/antagonists. Large libraries of compounds are needed for such screening. As a result of developments in this field, it is now possible to simultaneously produce combinatorial libraries containing hundreds of thousands of small molecules for screening. With the availability such libraries, however, has come a need for large scale, rapid screening methods.
The use of microtiter plates has significantly improved screening of combinatorial libraries. The plates allow assays to be miniaturized and have provoked the development of automated liquid handling and detection instruments to improve throughput and reproducibility. Robotics have allowed integration of compound arraying, microtiter plate handing, liquid handling, data acquisition and data processing. Even when facilitated by robotics, however, the steps of weighing, distributing, dissolving and serially diluting compounds in microtiter plates are time consuming.
The availability of combinatorial libraries on polymer beads have simplified these processes. By synthesizing sufficient compound on each bead for a few assays, compound handling is reduced or eliminated. Furthermore, thousands of beads can be arrayed in a given assay, and the compounds on the beads detached by photolysis, or other cleavage methods, into the assay in the concentration desired. Assaying library beads in standard microtiter plates therefore results in substantial improvement over the conventional assaying of bulk synthesized compounds.
The advantages of the bead format, however, have not been fully realized. In particular, scanning thousands of microtiter plate wells for positive results can be time consuming. Also, when beads are assayed in a microtiter well, only low concentrations of compounds are obtained since each bead contains only a small amount of compound. There is a need, therefore, for a more efficient method of screening large combinatorial libraries on beads, or other solid supports. There is also a need for a method which can assay higher concentrations of compounds on beads.
WO94/02515 describes screening a library of beads immobilized on a substrate, such as agarose. Molecules are partially cleaved from the beads and diffuse through the substrate, where they contact receptors on the surface of melanocytes. This causes pigment aggregation or dispersion within the cells, providing a signal that indicates where molecules have bound. WO94/02515 also describes determining immunological binding by diffusing labeled molecules in a gel, and contacting the molecules with antibodies on a plate below the gel, or in a second gel. Unbound molecules must be washed away, and the label detected. WO 94/02515 does not describe any method for detecting molecules that affect the activity of enzymes. It also does not describe any method for detecting binding to proteins without washing steps, which are particularly difficult to perform when using gels. In addition, it does not describe any method for screening compounds for binding to receptors that is not dependent on a biological response within a cell.